Glycohemoglobin is widely used as an indicator of a long-term glycemic control in diabetic patients. Glycohemoglobin in which glucose is nonenzymatically attached to hemoglobin is one of the glycated proteins. Among glycohemoglobin, particularly a fraction referred to as HbA1c is produced by binding glucose to an N-terminal valine residue of the β-chain in hemoglobin A to form a Schiff base aldimine (labile form) and then undergoing Amadori rearrangement to form a ketoamine. Glycohemoglobin with an aldimine structure is referred to as labile glycohemoglobin, whereas glycohemoglobin with a ketoamine structure is referred to as stable glycohemoglobin. After the Amadori rearrangement, the N-terminal of the β-chain becomes a fructosyl valine residue.
No enzyme is involved in this process, and its amount is increased depending on a glucose concentration in plasma. When so-called a blood glucose concentration in plasma is high in average for a long time, the HbA1c value becomes high. The stable glycohemoglobin does not disappear until a life span of an erythrocyte is terminated. It is generally known that the life span of a hemoglobin molecule in vivo is about 2 months, and that as a result, the HbA1c value reflects the average blood glucose concentration during the past one to two months. Thus, the HbA1c value is used as the indicator of the average blood glucose concentration over a long period of time. Generally, the blood glucose concentration changes easily, depending on the lifestyle type and diet before the diagnosis examination. However, the HbA1c value is the average value for a long time, and thus is suitable for using as information for making a decision for a definite diagnosis and a therapy of diabetes.
Thus, many of the various methods for assaying glycohemoglobin have been already proposed. As their representatives, high performance liquid chromatography (HPLC) methods, immunoassays and enzymatic assays are included.
The HPLC method is most frequently used at present. Hemoglobin is fractionated on a separation column, and a so-called area normalization method in which an abundance ratio of HbA1c is calculated from a peak area of an elution at a retention volume corresponding to HbA1c and a total peak area is employed. Thus, it is advantageous in that an accuracy of an injected volume can be ignored to some extent. However, it includes problems in that the apparatus is large and complicated, and its maintenance is time-consuming. Also, it has faults that the separation and measurement must be performed after previously removing labile HbA1c because labile HbA1c and stable HbA1c can not be distinguished (Patent Document 1). Furthermore, because of hemoglobin variants, a separation pattern occasionally varies to produce an abnormal value. It is also likely that obtained HbA1c value has errors because other biocomponents are accidentally overlapped with the peak of HbA1c.
The immunoassay has a possibility that the higher accurate determination is accomplished by a simpler system by taking advantage of an antibody against the structure in the vicinity of the N-terminus of the β-chain in HbA1c. However, differently from the general immunoassays, a sample hemolyzing whole blood is used instead of serum. Thus, nonspecific reactions easily occur and a calorimeter for the detection of the reaction is easily contaminated. Accordingly, it has been disclosed that satisfactory results are not always obtained.
Meanwhile, in the enzyme assays, a fructosyl peptide or a fructosyl amino acid is produced from the glycated protein by some means, and the resulting fructosyl peptide or fructosyl amino acid is detected and quantified using an enzyme such as fructosyl peptide oxidase or fructosyl amino acid oxidase. It is likely possible to carried out the higher accurate determination by taking advantage of the selectivity in the enzyme.
First, the means for determining the fructosyl amino acid by the enzyme is described in Patent Document 2, but the means for producing the fructosyl amino acid from the glycated protein is not described.
In addition, there are still many problems to be solved.
First, a protease which produces the fructosyl peptide or the fructosyl amino acid as rapidly as possible is required. At the same time, the protease with high activity likely digests not only hemoglobin but also fructosyl peptide oxidase and fructosyl amino acid oxidase. Thus, the method for the selective digestion of hemoglobin has been sought, but no effective method has been found yet.
Second, the N-terminal amino acids in both of the α-chain and the β-chain in hemoglobin are valine residues, and in the case of detecting the fructosyl amino acid, it is desirable that the protease which librates the fructosyl amino acid ideally acts upon only the β-chain. However, no protease with the high selectivity for extremely similar substrates such as the α-chain and the β-chain has been found yet.
From another standpoint, a solution has been proposed for this problem. That is, the fructosyl amino acid librated by the protease is not detected, and the glycated a chain and the glycated β chain are distinguished by detecting the fructosyl dipeptide or the fructosyl peptide. Particularly, an oxidase which selectively acts upon fructosyl valylhistidine in fructosyl dipeptides is proposed (Patent Documents 3, 4 and 5). By taking advantage of fructosyl peptide oxidase, which acts upon the fructosyl valylhistidine, it is possible to decrease requirements for the substrate specificity in the protease.
However, in Patent Document 3, the means for determining the glycated protein by measuring a sample digested with the protease using fructosyl peptide oxidase or HPLC has been disclosed. However, another specificity that the protease acts upon only the glycated protein and does not act upon fructosyl peptide oxidase has not been investigated. Even in Examples, only an example in which the protease is inactivated in heat and subjected to ultrafiltration is described.
In Patent Document 4, an enzyme activity is assayed using standard substrates of a low molecular weight, and the means for librating the fructosyl peptide from the glycated protein is not disclosed.
In Patent Document 5, the method for the determination of HbA1c by measuring the fructosyl peptide in the glycohemoglobin using the extremely excessive protease from Streptomyces sp. is exemplified. However, as described in Patent Document 5, many fructosyl peptide oxidases also have the ability to act upon fructosyl amino acid. Therefore, in the presence of extremely excessive protease, it is not clear which of the fructosyl peptide or the fructosyl amino acid is acted upon, and there is no example in which the specificity for the hemoglobin β-chain characteristic for fructosyl peptide oxidase has been completely exerted.
In Patent Document 6, the combination of the enzyme which acts upon the fructosyl peptide or the fructosyl amino acid to produce hydrogen peroxide with the protease producing its substrate was investigated. In this patent, the activity of the protease to produce the fructosyl amino acid is evaluated as the activity to liberate the fructosyl amino acid from the fructosyl dipeptide. The activity to produce the fructosyl dipeptide is evaluated as the activity to liberate a nitroaniline derivative from a nitroanilide derivative of the fructosyl dipeptide. Therefore, it is not clear whether the fructosyl peptide or the fructosyl amino acid is actually librated from glycated protein with high molecular weight, particularly glycohemoglobin. In fact, the inventors of the present application could not confirm the effectiveness of papain which was the protease from a plant and had been described to be effective in this invention.
In Patent Document 7, as the means for librating the fructosyl dipeptide, various proteases are exemplified, but actually, the means for librating fructosyl dipeptide from fructosyl hexapeptide has been investigated, and glycohemoglobin has not been digested. In a replication study by the inventor of the present application, no response was observed at all in the proteases, particularly papain and the protease from Aspergillus sp. except one from Bacillus sp., and the response was observed only in the protease from Bacillus sp. It was indicated that the replication study was not effected. A reason for this is not clear, but it is suggested that another product produced simultaneously with fructosyl valylhistidine by the proteolysis is detected in Patent Document 7.
When glycohemoglobin is consistently measured, it is necessary that the substrate used for the screening of the protease is hemoglobin itself. When a protease is utilized, it is desirable to detect only the fructosyl peptide. However, conventionally, the means for efficiently digesting glycohemoglobin and detecting only the required fructosyl peptide has not been disclosed.
Furthermore, no method and no apparatus which detects the total amount of hemoglobin in the sample and calculates the ratio of glycohemoglobin simply and accurately have been disclosed.    Patent Document 1: JP Hei-05-59380-B    Patent Document 2: JP Hei-05-33997-B    Patent Document 3: JP 2001-95598-A    Patent Document 4: JP 2003-235585-A    Patent Document 5: JP 2004-275013-A    Patent Document 6: JP 2004-344052-A    Patent Document 7: JP 2005-110657-A